Methods and materials for detection of biologicals

ABSTRACT

Methods of detecting biologicals in samples is provided herein. The detection is based on the formation of aggregates. The disclosed compositions include labeling particles and/or aggregating particles. The labeling particles and the aggregating particles may each include a receptor bound to the particle. The receptor can be either directly attached to the particle or indirectly attached to the particle through a linker. One method of detection may be visual and another may include advanced quantification of the formed aggregates.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 national phase application of PCTApplication No. PCT/US2014/026587, filed Mar. 13, 2014, which claimspriority to U.S. Provisional Application No. 61/784,820, filed Mar. 14,2013, each of which is incorporated herein by reference in its entirety.

FIELD

The present disclosure is directed to materials designed to detectbiologicals in fluids and on surfaces and to quantification of theirconcentrations by forming detectable and quantifiable aggregates, and tomethods of manufacturing and using the same.

BACKGROUND

Considerable resources are expended each year to measure and detectmicroorganisms and biologicals. Ensuring products, public areas, bloodand tissue banks, air and water supplies are free of microbialcontamination is a major public concern. The occurrence of microorganismand harmful biological contamination in any of these is a major healthrisk and detection and control thereof are a necessity.

Furthermore, there exists no assay for direct detection of bacteria insamples, such as body fluids, and for diagnosis of infections. Currentmethods instead diagnose indirectly, for example, by urinalysis, todetermine specifically the presence of nitrites, leukocytes, orleukocyte esterase and chemical testing for glucose or pH. Diagnosis ofan infection, if confirmed, is done so by bacterial culture, which canbe laborious and time consuming. Similarly, there is no direct test forbacteria in cerebrospinal fluid, which could be used for the rapiddiagnosis of bacterial meningitis, a rapidly progressing infection ofthe central nervous system, which is typically fatal if antibiotictreatment is not initiated promptly. Culture methods are too slow to beof use in diagnosing such infections.

Conventional detection methods may also be misleading and do not providerapid results allowing for immediate action. While current methods maybe effective tools to detect and quantify some types of microorganisms,most methods are culture-based or, if developed, molecular-basedtechniques for specific microorganisms and biologicals. However, thesemethods can be difficult and time-consuming. In addition, manymicroorganisms fail to have specific, reliable detection methods, suchas many viruses. Some microorganisms also may pose too hazardous forhandling, so no detection method is available.

Many tests exist for sensitive detection of a broad spectrum of variousbacterial species based on the detection of specific bacterial antigens.However, the tests are limited since they cannot be applied directly fortesting of samples where the spectrum of bacterial pathogens is unknown.Also, the tests include an additional step for pre-enrichment ofbiological. Such a step can take one-to-three days, before performingthe advertised tests. Thus, there remains a need for the development ofa detection method capable of detecting a broad spectrum ofmicroorganisms, known and unknown, that is easy to employ, fast andaccurate.

SUMMARY

The present disclosure provides methods of manufacturing and usinglabeling particles and formulations to detect biologicals in targetsamples. One method of detection may be visual as a result of theattachment of biologicals to labeling particles. The resultingbiological-particle complexes maybe physically separated from the sampleusing such methods as magnetic separation, filtration or decantation.The visual detection methods may be performed with a naked eye. Otherdetection methods can be used to qualify or quantify biologicals.

Some embodiments pertain to methods of detecting a biological includingthe steps of passing or mixing a sample comprising the biological overor with a labeling particle comprising a particle and receptor bound tothe particle and detecting the biological by adsorbing the biological tothe labeling particle. The particle can be of various sizes of, forexample, metallic nano-particles. The receptor can be any chemicalmoiety known or expected to bind to target biologicals. In one exemplaryembodiment, the composition can include a linker bridging between theparticle and the receptor. The linker is covalently bonded to theparticle and the receptor, and it can be a linear or branched moleculeor polymer. In addition, the target biological can be for example amicrobe, protein, or a blood product. A pre-enrichment of the targetbiological is not required.

Some embodiments pertain to methods of detecting a biological includingthe steps of passing or mixing a sample comprising the biological overor with a labeling particle and an aggregating particle, both comprisinga particle and receptor bound to the particle and detecting thebiological by adsorbing the biological to the labeling particle and tothe aggregating particle. One advantage of using the aggregatingparticle is to increase the size of the resulting biological-particleaggregate. The labeling particle and the aggregating particle cancontain various sizes of agarose, sand, textile, metallic particles,magnetic particles, or combinations thereof. The receptor in thelabeling particle and/or the aggregating particle can be any chemicalmoiety known or expected to bind to target biologicals. In one exemplaryembodiment, the composition of the labeling particle and/or theaggregating particle can include a linker bridging between the particleand the receptor. The linker is covalently or physically bonded to theparticle and the receptor, and it can be a linear or branched smallmolecule or polymer. The labeling particle and the aggregating particlecan contain the same or different linker and/or receptor. In addition,the target biological can be for example a microbe, protein, or a bloodproduct.

The methods of detecting a biological can also include visual detectionof the biological adsorbed to the labeling particle and/or aggregatingparticle as one method of detection. This visual detection can takeplace in the fluids or on the surfaces to be tested.

In some embodiments, the method of detecting a biological can includefiltering the biological adsorbed to the labeling and/or aggregatingparticle through a filtering setup as another method of detection. Insome embodiments, the method can include separating the biologicaladsorbed to the magnetic particle with a magnetic separator as anothermethod of detection.

The methods can also include qualifying or quantifying the biological,or combinations thereof, adsorbed to the labeling particle oraggregating particle, or combinations thereof, as one method ofdetection. The detection of the biological-particle aggregate isachieved with a naked eye or electronically, using tools, softwares andapplications capable of qualifying and quantifying colors and theirintensities. Such tools can include optical density measurement devicesand softwares and color intensity measurement devices and softwares.

The present disclosure also provides methods of manufacturing a labelingparticle and/or an aggregating particle including immobilizing areceptor on a particle. The receptor can be heparin or lactose.

BRIEF DESCRIPTION OF THE DRAWINGS

The appended drawings have been included herein so that theabove-recited features, advantages and objects of the disclosure willbecome clear and can be understood in detail. These drawings form a partof the specification. It is to be noted, however, that the appendeddrawings illustrate suitable embodiments and should not be considered tolimit the scope of the disclosure.

FIG. 1 illustrates the detection of biologicals, for example microbes,by forming light-scattering aggregates.

FIG. 2 illustrates three different embodiments: particles arefunctionalized with receptors either directly or indirectly throughlinkers.

FIGS. 3A-D illustrate examples of direct attachment of receptors toparticles.

FIGS. 4A-D illustrate examples of attachment of receptors to particlesvia linkers.

FIG. 5 illustrates a general route for covalent coupling when using1,1′-carbonyldiimidazole.

FIG. 6 illustrates a schematic representation of an application.

FIG. 7 illustrates aggregation of the labeling particles, functionalizedgold nanoparticles, with aggregating particles, functionalized sand.

FIG. 8 illustrates aggregation of the labeling particles, functionalizedgold nanoparticles, with aggregating particles, functionalizedpoly(glycidyl methacrylate) (PGMA).

FIG. 9 illustrates examples of applications of the disclosure; suchexamples are classified as qualitative biological detection methods.

FIG. 10 illustrates examples of applications of the disclosure; suchexamples are classified as quantitative biological detection methods.

FIG. 11 illustrates examples of applications of the disclosure; suchexamples are classified as combination of known aggregate detectionprocess with the invented process.

FIG. 12 illustrates examples of results showing a correlation betweenthe Absorbance of Escherichia coli bacteria aggregates and the titer ofEscherichia coli bacteria in the examined solutions.

DESCRIPTION

Point-of-use detection is performed at the place where a product or aservice is actually used. Major benefits are obtained when detection ofbiologicals is achieved at the point-of-use. Examples of such benefitsinclude limiting or avoiding exposure to infected fluids in remote orpoor areas where access to appropriate tests and detection methods isnot feasible, detecting infected supplies of blood and their productsprior to transfusion, and alerting to the presence of infectiousparticles in the air or on surfaces. Another example is diagnosingabnormal compositions of bodily fluids at the point-of-test and alertingthe patient to take actions against diseases immediately after theironset. Such early detection methods can speed recovery and reduce costsassociated with medical treatments.

Detection of biologicals using minimal materials and devices isextremely attractive for point-of-use diagnostics. Whilenanoparticle-based technologies may be useful, the two main approachesare based on antigen-antibody interactions and recognition of nucleicacids. Both techniques require expensive devices and reagents.

Certain exemplary embodiments will now be described to provide anoverall understanding of the principles of the structure, function,manufacture, and use of the devices and methods disclosed herein. One ormore examples of these embodiments are illustrated in the accompanyingdrawings. Those skilled in the art will understand that the devices andmethods specifically described herein and illustrated in theaccompanying drawings are non-limiting exemplary embodiments and thatthe scope of the present disclosure is defined solely by the claims. Thefeatures illustrated or described in connection with one exemplaryembodiment may be combined with the features of other embodiments. Suchmodifications and variations are intended to be included within thescope of the present disclosure.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contentclearly dictates otherwise. The terms used in this disclosure adhere tostandard definitions generally accepted by those having ordinary skillin the art. In case any further explanation might be needed, some termshave been further elucidated below.

The disclosed methods and materials allow visual detection of targetbiologicals. In the presence of labeling particles, e.g. functionalizednanoparticles, target biologicals can trigger the formation ofaggregates that absorb, reflect or scatter light, or any combinationthereof. Also, the optional presence of aggregating particles in theformulation composed of labeling particles and biological can accentuatethe signal that is due to absorption, reflection, or scattering oflight, or combinations thereof. Such smart materials may providecritical information on biologicals in fluids at the point-of-use.Ultimately, the intensity of the color of the aggregates can becorrelated with the concentration of specific biologicals. The methodcan be similar to “a litmus test for biologicals.”

The term “biologicals” as used herein refers to living organisms andtheir products, including, but not limited to, cells, tissues, tissueproducts, blood, blood products, proteins, vaccines, antigens,antitoxins, viruses, microorganisms, fungi, yeasts, algae, bacteria,etc. One example of a biological can include microorganisms, such aspathogenic or a non-pathogenic bacteria. Another example of biologicalscan include viruses, viral products, virus-imitating entities, or anycombination thereof.

A “microorganism” (i.e., a microbe) as used herein can be a single cellor multicellular organism and includes organisms such as prokaryotes(e.g., bacteria and archaea), eukaryotes (e.g., protozoa, fungi, algae,microscopic plants and animals), and viruses. For example, the bacteriacan be gram negative or gram positive. In some embodiments, themicroorganism is selected from Staphylococcus aureus, Streptococcus,Escherichia coli (E. coli), Pseudomonas 30 aeruginosa, Mycobacterium,adenovirus, rhinovirus, smallpox virus, influenza virus, herpes virus,human immunodeficiency virus (HIV), rabies, chikungunya, severe acuterespiratory syndrome (SARS), malaria, dengue fever, tuberculosis,meningitis, typhoid fever, yellow fever, ebola, Shigella, Listeria,Yersinia, West Nile virus, protozoa, fungi, Salmonella enterica, Candidaalbicans, Trichophyton mentagrophytes, poliovirus, Enterobacteraerogenes, Salmonella typhi, Klebsiella pneumonia, Aspergillusbrasiliensis, and methicillin resistant Staphylococcus aureus (MRSA), orany combination thereof.

The disclosed methods and materials can detect biologicals by formingvisible aggregates, such as those that are visible to the naked eye.Light scattering, absorption, reflection, or any combination thereof, oflabeling particles can be used to detect target biologicals. Theaggregating particles and other particles can also be used in lightscattering, absorbing, or reflecting formulations, or any combinationthereof, to detect target biologicals.

The present disclosure provides methods to produce labelingnanoparticles that exhibit physical differences between the presence andabsence and upon changes in the concentration of target biologicals.Such differences can be visually detected, such as with the naked eye,or can be qualified and quantified using tools, softwares andapplications capable of qualifying and quantifying colors and theirintensities. One example of labeling particles can include magnetic ormetallic nanoparticles, such as gold nanoparticles. The absence of thetarget biologicals may also inhibit the formation of such aggregates.

In one embodiment, “labeling particles” can bind to target biologicalsand form aggregates. In some embodiments, the resulting aggregates canscatter, absorb and/or reflect light for visual detection. In someembodiments, the aggregates can be separated by such physical separationtechniques as magnetic, filtration, decantation, or phase separationprocess, or any combination thereof. Other separation techniques mayalso be used that are known by one who is skilled in the art.

Some embodiments include “aggregating particles” to accentuate thesignal that results from the labeling particles bound to biologicals.The aggregating particles can be used to increase the size of theaggregate. Such increases can lead to easier detection and more accuratedifferentiation between different concentrations of biologicals (FIG.1). Examples of such aggregating particles can include materialcomplexes, like functionalized sand, functionalized agarose, andfunctionalized polymers that are insoluble in the examined fluid.

Examples of the three main components of the labeling and aggregatingparticles include: 1) particles, that can be of various sizes ofagarose, sand, textiles (such as cellulose/cotton, wool, nylon,polyester), metallic particles (including nanoparticles, such as goldnanoparticles or silver nanoparticles), magnetic particles (includingnanoparticles), glass, fiberglass, silica, wood, fiber, plastic, rubber,ceramic, porcelain, stone, marble, cement, biological polymers, naturalpolymers and synthetic polymers [such as Poly(glycidyl methacrylate)(PGMA)], or any combination thereof; 2) receptors, such as lactose(natural and synthetic) and its derivatives (such as sialyllactose),mono- and poly-saccharides, heparin and chitosan, or any combination ofmultiple types of particles; and 3) linkers, such as linear or branchedsmall molecule or polymer, such as poly(ethylene glycol) (PEG, e.g.multi-arm branched PEG-amines) and poly(ethyleneimine) (PEI, variousratios of primary:secondary:tertiary amine groups), dendrons anddendrimers (e.g. hyperbranched bis-MPA polyester-16-hydroxyl) andchitosan, or any combination thereof. Each of the materials mayincorporate the particle and the receptor components. However,incorporating the linker component is optional. Also, the labelingparticles can be formulations containing various types of particles,receptors and optional linkers.

The disclosed particles in the labeling and aggregating particles can befunctionalized with capturing groups (“receptors”), the structures ofwhich can be, or derived from, cellular receptors, antibodies, orsynthetically derived from available data describing interaction oftarget biologicals with soluble molecules. The receptors can be directlyattached to the particle (FIG. 2, Mode A) or through a linker (FIG. 2,Mode B). In order to protect the integrity of the molecular structure ofthe particle, particularly when reuse of the labeling and aggregatingparticle is a requirement, one method of inter-connecting the receptors,particles and, optionally linkers, is via covalent bonding. For certainapplications where added structural stability is not needed, for examplein case of single use materials, physical bonding can substitutecovalent bonding. The receptors play a direct role by capturing thebiologicals through physical bonding. One role of the linkers is toposition the receptors at an active distance from the core of theparticle. By distancing the receptors from the core of the particle, thereceptors can easily access the target biologicals. Another role for thelinkers, particularly when they are branched, is to increase the densityof the receptors on the surface of the particle (FIG. 2, Mode C).Increase in the density of receptors correlates with an increase in thecapacity of capturing higher concentrations of biologicals. In someembodiments, the receptor attached to the labeling particle and thereceptor attached to the aggregating particle are the same receptor(e.g., lactose molecules may be attached to the labeling particle and tothe aggregating particle). In some embodiments, the receptor attached tothe labeling particle and the receptor attached to the aggregatingparticle are different receptors (e.g., lactose molecules may beattached to the labeling particle and heparin molecules may be attachedto the aggregating particle).

Exemplary receptors in the labeling and aggregating particles caninclude: 1) heparin, a negatively charged polymer that mimics innateglycosaminoglycanes found in the membranes of host cells. It iscommercially available as heparin sodium which is extracted from porcineintestinal mucosa and is approved as blood anti-coagulant. Also,non-animal-derived synthetic heparin-mimicking sulfonic acid polymerscan act in a similar fashion to natural heparin; 2) chitosan, anecologically friendly bio-pesticide that can ligate to a variety ofmicro-organisms and proteins. It is also used as a hemostatic agent andin transdermal drug delivery; and 3) lactose, a by-product of the dairyindustry. It is widely available and produced annually in millions oftons. Lactose can also be synthesized by condensation/dehydration of thetwo sugars galactose and glucose, including all their isomers.

Exemplary particles in the labeling and aggregating particles caninclude metallic or magnetic particles, such as alkali, alkaline,transition, post-transition, lanthanide and actinide metal elements orcompounds. Some examples of metallic or magnetic elements or compoundsinclude, but are not limited to, lithium, sodium, potassium, rubidium,cesium, francium, beryllium, magnesium, calcium, strontium, barium,radium, aluminum, gallium, indium, tin, thallium, lead, bismuth,scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel,copper, zinc, yttrium, zirconium, niobium, molybdenum, technetium,ruthenium, rhodium, palladium, silver, cadmium, lanthanum, hafnium,tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, mercury,actinium, rutherfordium, dubnium, seaborgium, bohrium, hassium,meitnerium, darmstadtium, roentgenium, cerium, praseodymium, neodymium,promethium, samarium, europium, gadolinium, terbium, dysprosium,holmium, erbium, thulium, ytterbium, lutetium, thorium, protactinium,uranium, neptunium, plutonium, americium, curium, berkelium,californium, einsteinium, fermium, mendelevium, nobelium, andlawrencium, their derivatives, salts, and oxides, or any combinationthereof, including combinations to form magnetic compounds. In oneembodiment, gold nanoparticles can be the particles. In this embodiment,the smaller the size the nanoparticles is, the more red the reflectedcolor will be. Also in this embodiment, the larger the size of thenanoparticles, the reflected color will be more blue/purple.

Other exemplary particles in the labeling and aggregating particles caninclude, but are not limited to, glass, silica, sand, wood, fiber,natural and synthetic polymers, rubber, copper, gold, silver, platinum,zinc, stone, stainless steel, nickel, titanium, tantalum, aluminum,Nitinol, Inconel, iridium, tungsten, silicon, magnesium, tin, alloys,and coatings containing any of the foregoing, galvanized steel, hotdipped galvanized steel, electrogalvanized steel, annealed hot dippedgalvanized steel, or any combination thereof.

Glass materials suitable for use include, but are not limited to, sodalime glass, strontium glass, borosilicate glass, barium glass,glass-ceramics containing lanthanum, as well as any combination thereof.

Sand materials suitable for use include, but are not limited to, sandcomprised of silica (e.g., quartz, fused quartz, crystalline silica,fumed silica, silica gel, and silica aerogel), calcium carbonate (e.g.,aragonite), or any mixtures thereof. The sand can comprise othercomponents, such as minerals (e.g., magnetite, chlorite, glauconite,gypsum, olivine, garnet), metal (e.g., iron), shells, coral, limestone,rock, or any combination thereof.

Wood materials suitable for use include, but are not limited to, hardwood and soft wood, and materials engineered from wood, wood chips, orfiber (e.g., plywood, oriented strand board, laminated veneer lumber,composites, strand lumber, chipboard, hardboard, medium densityfiberboard). Types of wood include, but are not limited to, alder,birch, elm, maple, willow, walnut, cherry, oak, hickory, poplar, pine,fir, or any combination thereof.

Fiber materials suitable for use include, but are not limited to,natural fibers (e.g., derived from an animal, vegetable, or mineral) andsynthetic fibers (e.g., derived from cellulose, mineral, or polymer).Suitable natural fibers include, but are not limited to, cotton, hemp,jute, flax, ramie, sisal, bagasse, wood fiber, silkworm silk, spidersilk, sinew, catgut, wool, sea silk, wool, mohair, angora, and asbestos.Suitable synthetic fibers include, but are not limited to, rayon, modal,and Lyocell, metal fiber (e.g., copper, gold, silver, nickel, aluminum,iron), carbon fiber, silicon carbide fiber, bamboo fiber, seacell,nylon, polyester, polyvinyl chloride fiber (e.g., vinyon), polyolefinfiber (e.g., polyethylene, polypropylene), acrylic polyester fiber,aramid (e.g., TWARON™, KEVLAR™, or NOMEX™), spandex, or any combinationthereof.

Natural polymer materials suitable for use include, but are not limitedto, a polysaccharide (e.g., cotton, cellulose), shellac, amber, wool,silk, natural rubber, biopolymer (e.g., a protein, an extracellularmatrix component, collagen), or any combination thereof.

Synthetic polymer materials suitable for use include, but are notlimited to, polyvinylpyrrolidone, acrylics,acrylonitrile-butadiene-styrene, polyacrylonitrile, acetals,polyphenylene oxides, polyimides, polystyrene, polypropylene,polyethylene, poly(glycidyl methacrylate), polytetrafluoroethylene,polyvinylidene fluoride, polyvinyl chloride, polyethylenimine,polyesters, polyethers, polyamide, polyorthoester, polyanhydride,polysulfone, polyether sulfone, polycaprolactone, polyhydroxy-butyratevalerate, polylactones, polyurethanes, polycarbonates, polyethyleneterephthalate, as well as copolymers, or any combination thereof.

Rubber materials suitable for use include, but are not limited to,silicones, fluorosilicones, nitrile rubbers, silicone rubbers,polyisoprenes, sulfur-cured rubbers, butadiene-acrylonitrile rubbers,isoprene-acrylonitrile rubbers, and the like, or any combinationthereof.

Ceramic materials suitable for use include, but are not limited to,boron nitrides, silicon nitrides, aluminas, silicas, and the like, orany combination thereof.

Stone materials suitable for use include, for example, granite, quartz,quartzite, limestone, dolostone, sandstone, marble, soapstone,serpentine, or any combination thereof.

Exemplary linkers in the labeling and aggregating particles can include,but are not limited to: 1) chitosan, see its description as a receptor;2) linear or branched Poly(ethylene glycol) and their derivatives,produced from ethylene oxides and have many different chemical,biological, commercial and industrial uses; 3) linear or branchedpoly(ethyleneimine) (PEI, various ratios of primary:secondary:tertiaryamine groups); and 4) dendrons and dendrimers, relatively new molecules.They are repetitively branched molecules using a small number ofstarting reagents. They are commonly used in drug delivery and insensors.

In some embodiments, the particle at the core of the “labelingparticles” can be gold nanoparticles.

In some embodiments, receptors for simultaneous detection of mutipleknown and unknown microorganism can include heparin and its derivatives.

In some embodiments, the receptors can be directly attached to theparticle (FIG. 3) or through linkers via chemical coupling (FIG. 4). Onetype of coupling reagent is 1,1′-carbonyldiimidazole (CDI). However,others may be used such as N,N′-Dicyclohexylcarbodiimide (DCC) orN-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC orEDCI).

An exemplary coupling reagent is CDI. Basic protonated end groups, suchas surface-hydrolized gold, readily react with CDI to form an ester oramide link. The resulting imidazole-substituted derivatives are reactedwith hydroxyl-terminated receptors yielding either carbonates[R—O—C(O)—O-receptor] or carbamates [R—N(H)—C(O)—O-receptor]. Theresulting imidazole-substituted derivatives can also be reacted withamine-terminated receptors yielding urea derivatives[R—N(H)—C(O)—N(H)-receptor] (FIG. 5). Due to the formation of a covalentbond between the receptor and the particle (direct bonding or through alinker), the structure of the bound receptor is different compared tothe structure of the commercially available free receptor. For example,as depicted in FIG. 6, the receptor looses a hydrogen atom upon reactionwith the immidazole-substituted derivatives to form areceptor-carbonate, receptor-carbamate or receptor-urea derivative.

If an appropriate functional group is not present on the surface of theparticle, typically a suitable functional group can be made available onthe surface by a chemical transformation. In general, a chemicaltransformation can be hydrolysis, oxidation (e.g., using Collinsreagent, Dess-Martin periodinane, Jones reagent, and potassiumpermanganate), reduction (e.g., using sodium borohydride or lithiumaluminum hydride), alkylation, deprotonation, electrophilic addition(e.g., halogenation, hydrohalogenation, hydration), hydrogenation,esterification, elimination reaction (e.g., dehydration), nucleophilicsubstitution, radical substitution, or a rearrangement reaction. Ifneeded, more than one chemical transformation, successively orsimultaneously, can be used to provide a suitable functional group or aheterogeneous group of functional groups of various identities.Alternatively, a monomer with a desired functional group can be graftedto the material.

In some embodiments, the chemical transformation is hydrolysis.Generally, the hydrolysis is performed with water in the presence of astrong inorganic, organic, or organo-metallic acid (e.g., stronginorganic acid, such as hydrochloric acid, sulfuric acid, phosphoricacid, nitric acid, hydroiodic acid, hydrobromic acid, chloric acid, andperchloric acid) or strong inorganic, organic, or organo-metallic base(e.g., Group I and Group II hydroxides, such as lithium hydroxide,sodium hydroxide, potassium hydroxide, rubidium hydroxide, cesiumhydroxide, magnesium hydroxide, calcium hydroxide, and barium hydroxide;ammonium hydroxide; and sodium carbonate). For example, a materialcomprising an acyl halide can undergo hydrolysis to form a carboxylicacid.

In some embodiments, the chemical transformation is a substitutionreaction where one functional group is replaced with another. Forexample, a material comprising a haloalkyl group can react with a strongbase to form a hydroxy group.

In some embodiments, the chemical transformation is alkylation,hydrogenation, or reduction. For example, a material comprising ahydroxy or haloalkyl (e.g., iodoalkyl or bromoalkyl) moiety can bereacted with ammonia to form an amino group. A material comprising ahaloalkyl moiety also can be converted to a mercapto group byS-alkylation using thiourea. A material comprising a nitrile can behydrogenated to form an amino group. A material comprising an amidogroup can be reduced (e.g., in the presence of lithium aluminum hydride)to form an amino group. A material comprising a formyl or keto group canbe reduced to form an amino or hydroxy group. Multiple homogeneous orheterogeneous transformations can be applied simultaneously orsuccessively.

The labeling and aggregating particles can be formed by any suitablemethod using suitable temperatures (e.g., room temperature, reflux),reaction times, solvents, catalysts, and concentrations. In someembodiments, an excess amount of linkers and receptors will be used toensure an effective amount of receptors in the labeling and aggregatingparticles.

In some embodiments, attachments amongst receptors, linkers, andparticles can be secured physically. This is achieved by mixingreceptors or linkers, or combinations thereof, dissolved in solventswith the particles, then allowing the solvents to evaporate in air orunder vacuum.

In some embodiments, the particles can be mixed directly with the fluidto be tested, including fluid rinsate of solids and surfaces. Theparticles generally include the labeling particles. The inclusion of theaggregating particles is optional. When both types of particles areapplied, the mixing of particles with the fluid to be tested can besimultaneous, where both types of particles are mixed with the fluid atthe same time. It can also be successive, where one type of particle isfirst mixed with the fluid, followed by the other type.

An exemplary application method is a qualitative test to indicate thepresence or absence of target biologicals in the tested sample. Thefluid to be tested is directly mixed with the labeling particles. Aftermixing and optional incubation, the mixture is allowed to flow through abed of aggregating particles and/or a bed of filtering materials. Thepresence of target biologicals is indicated by the detection ofaggregates in the bed of aggregation particles and/or the bed offiltering materials (FIG. 9). The detection of the aggregates can beachieved by the naked eye and/or by more advanced techniques, such aselectronique techniques.

Another exemplary application method is a quantitative test to indicatethe titer of target biologicals in the tested sample. The titer can bezero or larger than zero. In addition to the fluid to be tested, samplescontaining known titers of target biologicals are processed in thisexemplary application. The fluid to be tested, containing unknown titerof target biological, and the fluids containing known titers aredirectly and separately mixed with the labeling particles. After mixingand optional incubation, the mixtures are allowed to flow through a bedof aggregating particles and/or a bed of filtering materials. Theaggregation level of the samples containing known titers can becorrelated with the known titers of the target biologicals. The presenceof target biologicals in the fluid to be tested is indicated by thedetection of aggregates in the bed of aggregation particles and/or thebed of filtering materials. Furthermore, the titer of the targetbiological is derived by comparing the aggregation level resulting fromthe fluid to be tested, to the aggregation levels of the samples ofknown titers (FIG. 10). The aggregation level of the samples can bequantified by the naked eye and/or by more advanced techniques, such aselectronique techniques such as optical density measurement, colorintensity quantification applications, softwares, and associated devicessuch as camera and camera phones (FIG. 11). A suitable method fordetecting and/or quantifying the aggregation level is to correlate thevalue of this level to the color intensity of the aggregate.

In some embodiments, the materials can be used for point-of-usebiological detection (FIG. 6). Biologicals in fluids, including air andother gases, can be detected and quantified. Examples of fluids caninclude, but are not limited to, water, body fluids, saliva, vomit,urine, tear, sweat, bile, breast milk, cerebrospinal fluid, feces,diarrhea, body ejaculate, body secretion, pus, mucus, lymph, gastricacid and juice, earwax, blister, humour, intracellular and extracellularfluids, blood and blood products, drinking water, waste water,industrial water, house water, plant fluids, and air.

In some embodiments, the materials can be used for point-of-usebiological detection in air and other gases. Biologicals in air andother gases can be detected and quantified. One example of a method ofbiological detection in air is to rinse a surface that is in directcontact with the air or gas to be examined, with a fluid. After that,applying the methods described herein to examine the biologicals in theresulting fluid rinsate. In one example, the surface can be the airfilter of an air-conditioning (cooling or heating) devices and systemsin such environments as hospitals, malls, hotels, airplanes, boats,trains, airports, seaports, boarders, markets. In another examples, thesurface can be the air filter of a breathing mask.

In some embodiments, the materials can be used for point-of-usebiological detection on solids and surfaces. Biologicals on solidsurfaces can be detected and quantified. One example of a method ofbiological detection on solid surface is to rinse the surface to beexamined with a fluid. After that, applying the methods described hereinto examine the biologicals in the resulting fluid rinsate. Examples ofcoated and un-coated solid surfaces can include, or derived from anysuitable form of, such as, for example, a powder, dust, an aggregate, anamorphous solid, a sheet, a fiber, a tube, a fabric, or the like. Insome embodiments, the solid surface can comprise, without being limitedto, metal, glass, fiberglass, silica, sand, wood, fiber, naturalpolymer, synthetic polymer, plastic, rubber, ceramic, porcelain, stone,marble, cement, human or animal body, plants, food, fruit, vegetable, ormeat, or any combination thereof. In other embodiments, the solidsurface can comprise, without being limited to, sheet, textile,laboratory coats, coats, gloves, curtains, door handles, tables, phones,railings, keyboards, computers, screens, bottoms, elevator bottoms,etc., or any combination thereof.

The present disclosure is also directed to compositions comprising oneor more of the labeling particles described herein and/or any one ofmore of the aggregating particles described herein, as well as kitscomprising any one or more of the compositions disclosed herein, and/orreagents used for making and/or using them. In some embodiments, thecompositions comprise one or more of the labeling particles describedherein. In some embodiments, the compositions comprise any one of moreof the aggregating particles described herein. In some embodiments, thecompositions comprise any one or more of the labeling particlesdescribed herein and any one of more of the aggregating particlesdescribed herein.

The receptors may also reversibly interact with the target biologicals,such as micro-organisms or virus. The biologicals can be desorbed fromthe receptors, such as through elution. Eluents such ashigher-then-physiological sodium chloride solutions andlactose-containing solutions are capable of desorbing the biologicalfrom the aggregating and labeling particles. U.S. ProvisionalApplication Ser. No. 61/784,820 filed Mar. 14, 2013 is incorporatedherein by reference in its entirety.

EXAMPLES Example 1 Synthesis of Chitosan-PGMA (FIG. 3A)

The synthesis followed these steps: Four hundred ml of 0.5% acetic acidin de-ionized (DI) water were prepared by adding 2 g of the acid to 400mL of water. To this acid solution, 2 g of low molecular weight chitosanwere added and the solution was allowed to stir at room temperature for5 minutes until it became monophasic. Then, 200 mg of PGMA was added andthe final suspension was allowed to stir at room temperature for twohours. The final off-white suspension was then filtered through a mediumfrit and the solid was washed with 100 ml of DI water. The isolated wetsolid was re-suspended in 10 ml DI water. Its pH was ca. 4. One drop ofa sodium carbonate solution (5 wt % sodium carbonate solution wasprepared by dissolving 500 mg of Na₂CO₃ in 9.5 g DI water) was added toincrease the pH to ca. 9. The product was isolated, rinsed with 50 ml DIwater, and its wetness was preserved.

Example 2 Synthesis of Heparin-Sepharose® (FIG. 3B)

The synthesis followed these steps: 0.25 g of Sepharose® (5 wt % inwater) was loaded into a 20 ml glass vial along with a stirring bar. Tenml of pH 8.5 20 mM borate buffer was added to the solid, followed by 5mg 1,1′-CDI. The mixture was allowed to stir for two hours and a halfbefore the addition of 12.5 mg heparin sodium. The final mixture wasallowed to stir for three days at room temperature. The product wasisolated, rinsed with 25 ml DI water, and its wetness was preserved.

Example 3 Synthesis of Heparin-Sand (FIG. 3C)

The synthesis followed these steps: 1 g of sand was loaded into a 20 mlglass vial along with a stirring bar. Ten ml of pH 8.5 20 mM boratebuffer was added to the solid, followed by 2.5 mg 1,1′-CDI. The mixturewas allowed to stir for two hours and a half before the addition of 6.25mg heparin sodium. The final mixture was allowed to stir for three daysat room temperature. The product was isolated, rinsed with 50 ml DIwater, and its wetness was preserved.

Example 4 Synthesis of Heparin-PGMA (FIG. 3D)

The synthesis followed these steps: First was the preparation ofPGMA-NH₂: Fifty ml dry tetrahydrofuran was added to a round bottomflask, followed by 1.24 g of 1,4-diaminobutane. While stirring thesolution, 200 mg PGMA were added. The solution was then allowed to stirat room temperature for 10 minutes before evacuation. To the resultingproduct, 50 ml DI water was added leading to the precipitation of asolid. This solid was then filtered on a medium frit and rinsed with 300ml DI water. The following step included the preparation ofheparin-PGMA: 0.05 g of PGMA-NH₂ was loaded into a 20 ml glass vialalong with a stirring bar. Ten ml of pH 8.5 20 mM borate buffer wasadded to the solid, followed by 10 mg 1,1′-CDI. The mixture was allowedto stir for two hours and a half before the addition of 25 mg heparinsodium. The final mixture was allowed to stir for three days at roomtemperature. The final product was isolated and its wetness waspreserved.

Example 5 Synthesis of Heparin-[Branching]-Sand (FIG. 4A)

The synthesis followed these steps: One gram of fine sand was mixed with10 ml pH 8.5 20 mM Borate buffer and allowed to stir for a few minutesat room temperature. Eight mg of 1,1′-carbonyldiimidazole was then addedto the suspension and allowed to stir for 2 hours and a half beforeadding 0.125 g of hyperbranched bis-MPA polyester-16-hydroxyl. After anadditional two hours and a half, 5 mg of 1,1′-carbonyldiimidazole wasadded to the suspension and allowed to stir for 2 hours before adding12.5 mg of heparin sodium. The final mixture was allowed to stir forthree days at room temperature. The final product was isolated and itswetness was preserved.

Example 6 Synthesis of Heparin-[Branching]-Sepharose® (FIG. 4B)

The synthesis followed these steps: Two hundred and fifty mg ofSepharose® (5 wt % in water) was mixed with 10 ml pH 8.5 20 mM Boratebuffer and allowed to stir for few minutes at room temperature. Sixteenmg of 1,1′-carbonyldiimidazole was then added to the suspension andallowed to stir for 2 hours before adding 0.25 g of hyperbranchedbis-MPA polyester-16-hydroxyl. After two hours and a half, 10 mg of1,1′-carbonyldiimidazole was added to the suspension and allowed to stirfor 2 more hours before adding 25 mg of heparin sodium. The finalmixture was allowed to stir for three days at room temperature. Theproduct was isolated, rinsed with 25 ml DI water, and its wetness waspreserved.

Example 7 Synthesis of Heparin-[Branching]-PGMA (FIG. 4C)

The synthesis followed these steps: Fifty mg of PGMA-NH₂ was mixed with10 ml pH 8.5 20 mM Borate buffer and allowed to stir for a few minutesat room temperature. Thirty two mg of 1,1′-carbonyldiimidazole was thenadded to the suspension and allowed to stir for 1 hour before adding 0.5g of hyperbranched bis-MPA polyester-16-hydroxyl. After two additionalhours, 20 mg of 1,1′-carbonyldiimidazole was added to the suspension andallowed to stir for 2 more hours before adding 50 mg of heparin sodium.The final mixture was allowed to stir for three days at roomtemperature. The product was isolated, rinsed with 25 ml DI water, andits wetness was preserved.

Example 8 Synthesis of Heparin-[Branching]-Gold Nanoparticles (FIG. 4D)

The synthesis followed these steps: Five ml of gold nanoparticlesolution in PBS was mixed with 2 ml pH 8.5 20 mM Borate buffer andallowed to stir for 30 minutes at room temperature in a glass vial.Eight mg of 1,1′-carbonyldiimidazole (0.05 mmol, MW 162.15) was thenadded to the suspension and allowed to stir for 2 hours before adding0.133 g of hyperbranched bis-MPA polyester-16-hydroxyl. After that, 20mg of 1,1′-carbonyldiimidazole was added to the suspension and allowedto stir for 2 hours before adding 50 mg of heparin sodium. The finalmixture was allowed to stir overnight at room temperature.

Example 9 Synthesis of Heparin-[Branching]-Sand

The synthesis will follow these steps: One gram of fine sand will bemixed with 10 ml pH 8.5 20 mM Borate buffer and allowed to stir for afew minutes at room temperature. Eight mg of 1,1′-carbonyldiimidazolewill then be added to the suspension and allowed to stir for 2 hours anda half before adding branched poly(ethyl glycol) (1.14 mmol.eq. OH).After an additional two hours and a half, 5 mg of1,1′-carbonyldiimidazole will be added to the suspension and allowed tostir for 2 hours before adding 12.5 mg of heparin sodium. The finalmixture will be allowed to stir for three days at room temperature. Thefinal product will be isolated and its wetness will be preserved.

Example 10 Synthesis of Heparin-[Branching]-Sepharose®

The synthesis will follow these steps: Two hundred and fifty mg ofSepharose® (5 wt % in water) will be mixed with 10 ml pH 8.5 20 mMBorate buffer and allowed to stir for a few minutes at room temperature.Sixteen mg of 1,1′-carbonyldiimidazole will then be added to thesuspension and allowed to stir for 2 hours before adding branchedpoly(ethylene glycol) (2.28 mmol.eq. OH). After two hours and a half, 10mg of 1,1′-carbonyldiimidazole will be added to the suspension andallowed to stir for 2 more hours before adding 25 mg of heparin sodium.The final mixture will be allowed to stir for three days at roomtemperature. The product will be isolated, rinsed with 25 ml DI water,and its wetness will be preserved.

Example 11 Synthesis of Heparin-[Branching]-PGMA

The synthesis will follow these steps: Fifty mg of PGMA-NH₂ will bemixed with 10 ml pH 8.5 20 mM Borate buffer and allowed to stir for afew minutes at room temperature. Thirty two mg of1,1′-carbonyldiimidazole will then be added to the suspension andallowed to stir for 1 hour before adding branched poly(ethylene glycol)(4.56 mmol.eq. OH). After two additional hours, 20 mg of1,1′-carbonyldiimidazole will be added to the suspension and allowed tostir for 2 more hours before adding 50 mg of heparin sodium. The finalmixture will be allowed to stir for three days at room temperature. Theproduct will be isolated, rinsed with 25 ml DI water, and its wetnesswill be preserved.

Example 12 Synthesis of Heparin-[Branching]-Gold Nanoparticles

The synthesis will follow these steps: Five ml of gold nanoparticlesolution in PBS will be mixed with 2 ml pH 8.5 20 mM Borate buffer andallowed to stir for 30 minutes at room temperature in a glass vial.Eight mg of 1,1′-carbonyldiimidazole (0.05 mmol, MW 162.15) will then beadded to the suspension and allowed to stir for 2 hours before addingbranched poly(ethylene glycol) (1.14 mmol.eq. OH). After that, 20 mg of1,1′-carbonyldiimidazole will be added to the suspension and allowed tostir for a 2 hours before adding 50 mg of heparin sodium. The finalmixture will be allowed to stir overnight at room temperature.

Example 13 Infected Surface Water

One hundred μl of surface water (non-potable but clear) from Lexington,Mass., was mixed with 50 μl of the heparin-[branching]-goldnanoparticles solution. The resulting solution was designated assolution A. 100 μl of potable clean water was mixed with 50 μl of theheparin-[branching]-gold nanoparticles solution. The resulting solutionwas designated as solution B. Both solutions, A and B, were mixed. TwoPasteur glass pipettes were stopped with cotton followed byheparin-[branching]-sand. These pipettes were then loaded with eithersolution A or B. The solutions were allowed to sit on sand for 10minutes before slowly forcing them through the sand beds. The cottonlayer in column A, where solution A was loaded, showed a dark red band,while the cotton layer in column B, where solution B was loaded, did notshow a dark red band (Examples in FIG. 7).

Example 14 Infected Surface Water

One hundred μl of surface water (non-potable but clear) from Lexington,Mass., was mixed with 50 μl of the heparin-[branching]-goldnanoparticles solution. The resulting solution was designated assolution C. One hundred μl of potable clean water was mixed with 50 μlof the heparin-[branching]-gold nanoparticles solution. The resultingsolution was designated as solution D. Both solutions, C and D, weremixed. To each of the solutions, C and D, 5 mg ofheparin-[branching]-PGMA was added and allowed to mix. Two Pasteur glasspipettes were stopped with cotton. Solution C was loaded into the firstpipette and solution D was loaded into the second pipette. The solutionswere allowed to slowly migrate through the pipettes. The outcome bed inthe first pipette, where solution C was loaded, showed a dark red tobrown band, while the outcome bed in the second pipette, where solutionD was loaded, did not show this dark band (Examples in FIG. 8).

Example 15 Infected Surface Water

One hundred μl of surface water (non-potable but clear) from Lexington,Mass., was mixed with 50 μl of the heparin-[branching]-goldnanoparticles solution. The resulting solution was designated assolution E. One hundred μl of potable clean water was mixed with 50 μlof the heparin-[branching]-gold nanoparticles solution. The resultingsolution was designated as solution F. Both solutions, E and F, weremixed. To each of the solutions E and F, 5 mg of heparin-Sepharose® wasadded and allowed to mix at 200 rpm for 5 minutes. Two Pasteur glasspipettes were stopped with cotton. Solution E was loaded into the firstpipette and solution F was loaded into the second pipette. The solutionswere allowed to slowly migrate through the pipettes. Difference incoloration was observed between the two beds in the first and the secondpipette.

Example 16 Aqueous Solutions of Known Bacteria Titers

Preparation of the Escherichia coli (E. coli) inoculum: Lysogeny broth(LB) (1 mL) was added to a 15 ml cell culture tube. Seven (7) μl of 40%glycerol stock of the ONE SHOT™ TOP10 E. coli cells (Life Technologies,Grand Island, N.Y.) was inoculated into the 1 ml LB broth. The inoculatewas incubated at 37° C. for 3 hours with constant shaking (ca. 250 rpm).The inoculate was then centrifuged at 2500 rpm speed for 4 minutes. TheLB broth was separated, and the E. coli cells were washed twice with 1ml of sterile phosphate buffered saline (PBS) 1× (pH 7.4) each time andthen re-suspended in 1 mL of the same buffer. Using optical densitymeasurements, the final bacterium titer was estimated to be ca. 5×10⁷cells/ml. This solution was labeled Solution 84-1. Ten μl of Solution84-1 was mixed with 990 μl of sterile PBS 1×. The resulting bacteriumtiter was estimated at 5×10⁵ cells/ml. This solution was labeledSolution 84-2. Ten μl of Solution 84-2 was mixed with 990 μl of sterilePBS 1×. The resulting bacterium titer was estimated at 5×10³ cells/ml.This solution was labeled Solution 84-3. Ten μl of Solution 84-3 wasmixed with 990 μl of sterile PBS 1×. The resulting bacterium titer wasestimated at 50 cells/ml. This solution was labeled Solution 84-4.

Example 17 Aqueous Solutions of Known Bacteria Titers

Correlation of the Escherichia coli (E. coli) titer with the colorintensity of the formed aggregates: 200 μl of each of the aboveSolutions 84-1, 84-2, 84-3, 84-4 were mixed with 25 μl ofheparin-[branching]-gold nanoparticles solution. The resulting solutionswere passed through 96-well filter plates (Millipore MAFCNOB). TheAbsorbance values in these wells were then read using a Tecan platereader (FIG. 12). Absorbance reading (Abs.) at 505 nm wavelengthrevealed the following values: 1) for Solution 84-1, Abs. 1.9505; 2) forSolution 84-2, Abs. 1.8965; 3) for Solution 84-3, Abs. 1.8829; 4) forSolution 84-4, Abs. 1.8527. Absorbance reading (Abs.) at 515 nmwavelength revealed the following values: 1) for Solution 84-1, Abs.1.9434; 2) for Solution 84-2, Abs. 1.8853; 3) for Solution 84-3, Abs.1.8764; 4) for Solution 84-4, Abs. 1.8482. Absorbance reading (Abs.) at525 nm wavelength revealed the following values: 1) for Solution 84-1,Abs. 1.9373; 2) for Solution 84-2, Abs. 1.8750; 3) for Solution 84-3,Abs. 1.8685; 4) for Solution 84-4, Abs. 1.8363. Absorbance reading(Abs.) at 535 nm wavelength revealed the following values: 1) forSolution 84-1, Abs. 1.9202; 2) for Solution 84-2, Abs. 1.8646; 3) forSolution 84-3, Abs. 1.8537; 4) for Solution 84-4, Abs. 1.8288.Absorbance reading (Abs.) at 545 nm wavelength revealed the followingvalues: 1) for Solution 84-1, Abs. 1.9125; 2) for Solution 84-2, Abs.1.8573; 3) for Solution 84-3, Abs. 1.8475; 4) for Solution 84-4, Abs.1.8209.

The invention claimed is:
 1. A method for detecting a biologicalcomprising: mixing a sample with a plurality of particles each of whichcomprises a receptor adapted for binding to the biological such thatsaid particles form one or more aggregates if said biological is presentin the sample and binds to the receptor; and identifying presence of thebiological in the sample by detecting one or more aggregates of saidparticles, wherein at least one of the particles further comprises acovalently bonded linker molecule, and wherein the receptor iscovalently bonded to the particle via said linker molecule, and whereinthe linker molecule is selected from the group consisting of branchedpoly(ethylene glycol) (PEG), branched poly(ethyleneimine) (PEI),hyperbranched bisMPA polyester-16-hydroxyl, and any combination thereof.2. The method of claim 1, wherein at least one of said particles is anyof a light absorbing particle, a reflecting particle, a scatteringparticle, a metallic particle, a magnetic particle, or any combinationthereof.
 3. The method of claim 1, wherein the receptor is selected fromthe group consisting of at least one lactose, lactose derivative, mono-or poly-saccharide, heparin, or chitosan, or any combination thereof. 4.The method of claim 1, wherein at least one of the particles is anabsorbing, reflecting or scattering particle, metallic particle,magnetic particle, agarose, polysaccharide, natural sand, processedsand, textile, cellulose, cotton, wool, nylon, polyester, nanoparticle,glass, fiberglass, silica, wood, fiber, plastic, rubber, ceramic,porcelain, stone, marble, cement, biological polymer, natural polymer,synthetic polymer, or any combination thereof of various particle sizes.5. The method of claim 4, wherein the metallic particle is a goldparticle or nanoparticle, or a silver particle or nanoparticle, or acombination thereof; and wherein the synthetic polymer is Poly(glycidylmethacrylate) (PGMA), or PGMA-NH₂, and the natural polymer is cotton, orany combination thereof.
 6. The method of claim 1, further comprisingseparating said one or more aggregates from the sample using any offiltration, decantation, and magnetism, or any combination thereof. 7.The method of claim 1, wherein the biological is selected from the groupconsisting of cell, tissue, tissue product, blood, blood product, bodyfluid, product of body fluid, protein, vaccine, antigen, antitoxin,biological medicine, biological treatment, virus, microorganism, fungus,yeast, alga, bacterium, prokaryote, eukaryote, Staphylococcus aureus,Streptococcus, Escherichia coli (E. coli), Pseudomonas aeruginosa,Mycobacterium, adenovirus, rhinovirus, smallpox virus, influenza virus,herpes virus, human immunodeficiency virus (HIV), rabies virus,chikungunya virus, severe acute respiratory syndrome (SARS) virus, poliovirus, malaria parasite, dengue fever virus, tuberculosis bacterium,meningitis microorganism, typhoid fever bacterium, yellow fever virus,ebola virus, Shigella, Listeria, Yersinia, West Nile virus, protozoa,fungi, Salmonella enterica, Candida albicans, Trichophytonmentagrophytes, poliovirus, Enterobacter aerogenes, Salmonella typhi,Klebsiella pneumoniae, Aspergillus brasiliensis, and methicillinresistant Staphylococcus aureus (MRSA), or any combination thereof. 8.The method of claim 1, wherein the sample is a fluid selected from thegroup consisting of water, juices, fluid extracts, body fluids, saliva,vomit, urine, tear, sweat, bile, milk, breast milk, cerebrospinal fluid,feces, diarrhea, body ejaculate, body secretion, pus, mucus, lymph,gastric acid and juice, earwax, blister, humoral fluid, intracellularand extracellular fluids, blood and blood products, drinking water,waste water, industrial water, house water, plant fluids, rinsates ofsolid and surface, rinsates of food, rinsates of fruits, rinsates ofvegetables, air, and gas, or any combination thereof; or wherein thesample is a solid and/or surface thereof selected from the groupconsisting of coated and un-coated powder, dust, aggregate, amorphoussolid, crystalline solid, sheet, fiber, tube, fabric, metal, glass,fiberglass, silica, sand, wood, fiber, natural polymer, syntheticpolymer, plastic, rubber, ceramic, porcelain, stone, marble, cement,human or animal body, plant, food, fruit, vegetable, meat, textile,coat, glove, curtain, door handle, table, phone, railing, keyboard,computer, screen, button, or any combination thereof.
 9. The method ofclaim 1, wherein the detection of said one or more aggregates of theparticles is performed via visual inspection.
 10. The method of claim 1,wherein the detection of said one or more aggregates of the particles isperformed via color density measurement, optical density measurement, ora combination thereof.
 11. The method of claim 1, further comprisingquantifying aggregation level of said particles in the sample.
 12. Themethod of claim 11, wherein said step of quantifying the aggregationlevel of said particles is performed by any of visual inspection,optical density measurement, and color intensity quantification.
 13. Themethod of claim 11, further comprising comparing the aggregation levelof the particles in the sample with aggregation level of one or moresamples of known titers to obtain concentration of the biological in thesample.
 14. The method of claim 1, wherein a first plurality of theparticles is bound to one receptor type and a second plurality of theparticles is bound to a different receptor type.